Carbon monoxide releasing rhenium compounds for medical use

ABSTRACT

The present invention relates to new rhenium compounds of formula (I) 
     
       
         
         
             
             
         
       
     
     with medical utility, corresponding pharmaceutical compositions as well as medical uses thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. Ser. No. 13/583,390, filed Sep.7, 2012, which is the U.S. national stage application of InternationalPatent Application No. PCT/EP2011/001077, filed Mar. 4, 2011.

FIELD OF THE INVENTION

The present invention relates to new rhenium compounds, preferablynon-radioactive rhenium compounds for medical use, correspondingpharmaceutical compositions as well as medical uses thereof.

BACKGROUND OF THE INVENTION

In recent years carbon monoxide has been acknowledged as a fundamentalsmall molecule messenger in mammals. The endogenous production of CO isassociated with the heme metabolic pathway, in particular the action ofa family of enzymes known as heme oxigenases, which catalyze theoxidation of heme to biliverdin and thereby liberate free iron andcarbon monoxide. The tissue-specific distribution of heme oxigenasesand, thus, the liberated carbon monoxide have been linked to severalphysiological effects. For example, carbon monoxide is a signalingmolecule in the inducible defensive system against stressful stimuli; ithas a fundamental role in the circulatory system by improvingvasorelaxation and cardiac blood supply; it suppresses arterioscleroticlesions associated with chronic graft rejection; like NO it influencesneurotransmission in the hypothalamic-pituitary-adrenal axis and thereis evidence that carbon monoxide influences the circadian rhythm ofmammals by interacting with NPAS2, the so-called human “clock” protein.

Due to the relevance of carbon monoxide for mammalian physiology, theinterest in its medicinal use is growing. Direct carbon monoxideinhalation was tested, but low tolerance to the compound provedcontradictory because there is a delicate balance between carbonmonoxide-induced tissue hypoxia and therapeutic benefits. Furthermore,the direct gaseous use leads to problems with safety as well as withtargeted and controlled delivery. CO-releasing molecules (CO-RMs)represent an alternative approach to the administration of carbonmonoxide. A number of complexes have been evaluated and pioneering workof Motterlini and Mann (e.g. Motterlini et al., Circ. Res., 2002 90,E17-24; Motterlini et al., Intensive Care Med. 2008, 34, 649-658;Motterlini et al., Circ. Res. 2002, 90, E17-24; Motterlini et al.,Expert. Opin. Investig. Drugs 2005, 14, 1305-1318; Motterlini et al.,Curr. Pharm. Des. 2003, 9, 2525-2539; Motterlini et al., J., FASEB J.2005, 19, 284-286) resulted in the most promisingfac-[RuCl(glycinato)(CO)₃] complex (CO-RM-3) for controlled carbonmonoxide release in vivo. The chemistry and therapeutic effects ofCORM-3 are well-documented. CORM-3 releases one mol carbon monoxidewithin ten minutes after being dissolved in water and significantlyreduces blood pressure in vivo and relaxes pre-contracted aortic ringsin vitro. Its cardioprotective effects have been documented. Today metalcarbonyls have been recognized as a potential new class ofpharmaceuticals (for a review on CO-RMs see Johnson et al., MetalCarbonyls in Medicine, Angew. Chem. Int. Ed., 2003, 42, 3722-3729).There are a wide range of documented physiological and medicallybeneficial effects of CO. It is anti-inflammatory, e.g. it attenuatesendotoxic shock and reduces allergic inflammation; it suppresses graftrejection; it protects against hyperoxia and oxidative lung injury; itprotects against ischemia and reperfusion injury; it protects pancreaticbeta cells from apoptosis; it modulates spermatogenesis under stressconditions; it decreases perfusion pressure; it protects against septicshock and lung injury; it provides cytoprotective effects; it modulatesvascular smooth muscle tone, regulates blood pressure under stressconditions and suppresses arteriosclerotic lesions associated withchronic graft rejection and with balloon injury. For CO-RMs aorticvasodilatation, attenuated coronary vasoconstriction and reduction ofhypertension was shown. For CO-RM3 inhibition of reperfusion injury,graft rejection and blood platelet aggregation was confirmed.

WO 02/092075 A2 pharmaceutical compositions comprising metal carbonylcompounds teaches, wherein the metal is selected from Fe, Mn, Ru, Rh,Ni, Mo or Co, for stimulating guanylate cyclase activity,neurotransmission or vasodilatation, for treating hypertension,radiation damage, endotoxic shock, inflammation, inflammation-relateddiseases, hyperoxia-induced injury, apoptosis, cancer, transplantrejection, arteriosclerosis, post-ischemic organ damage, myocardialinfarction, angina, haemorrhagic shock, sepsis, penile erectiledysfunction and adult respiratory distress symptom.

Rhenium is an extremely rare group 7 transition metal that is mainlyused for superalloy and catalyst production. Radioactive isotopes ¹⁸⁸Reand ¹⁸⁶Re are presently used for treatment of liver cancer. They bothhave similar penetration depths in tissue (5 mm for ¹⁸⁶Re and 11 mm for¹⁸⁸Re), but ¹⁸⁶Re has the advantage of longer lifetime (90 hours vs. 17hours for the 188 isotope). Related by periodic trends rhenium has achemistry similar to technetium. Results on work done to label rheniumonto target compounds can often be transferred to technetium. Thistransfer aspect has proven useful for radiopharmacy, where it isdifficult to work with technetium, especially with the medically used99m isotope. So far medical applications of rhenium have been limited tothe cytotoxic use of its radioactive isotopes.

Very recently Zobi et al. (Inorg. Chem. 2009, 48, 8965-8970) reportedthe synthesis of carbon monoxide-containing rhenium I and II complexesby means of a versatile synthetic intermediate [Re^(II)Br₄(CO)₂]²⁻, thatis stable but sufficiently sensitive to substitution reactions withselected ligands. Due to the unexpected aerobic stability andwell-behaved chemistry of complexes derived from this intermediate, theauthors very generally speculated on possible medical applications ofthese Re^(I/II) complexes in medicinal chemistry in the context ofconventional Re use, i.e. as cytotoxic isotopes.

It is the object underlying the present invention to provide newmedically useful rhenium compounds, preferably medically useful andnon-radioactive rhenium compounds. Furthermore, it is an object toprovide new carbon monoxide-releasing compounds for medical and/ordiagnostic applications, in particular for the prophylaxis and/ortreatment of diseases and/or medical conditions selected from the groupconsisting of cardiovascular diseases, preferably cardiac hypoxia,cardiac infarction, cardiac hypertrophy, arteriosclerosis andhypertension; ischemia-reperfusion injury, inflammatory diseases,preferably asthma or angina; traumatic injury, preferably of the brain,kidney or liver; transplant rejection, preferably allograft andxenograft rejection, platelet aggregation and/or monocyte activation;neuron degeneration of the nervous system, radiation damage, cancer,penile erectile dysfunction, adult respiratory distress syndrome, anddisorders of the circadian rhythm of mammals, preferably jet lag.

A further object is to provide (i) new diagnostic and/or medical usesfor rhenium compounds, in particular non-radioactive rhenium compoundsas well as (ii) pharmaceutical and diagnostic compositions comprisingsaid compounds.

SUMMARY OF THE INVENTION

In a first aspect, the above objects are solved by new compounds offormula (I), preferably for medical use:

wherein Re is Re(I) or Re(II), preferably Re(II),n is selected from 3− to 3+, preferably 2−, 1−, 0, 1+ and 2+, morepreferably 1−, 0, 1+;L1, L2, L3 and L4 denote in each case independently of one anotherpharmaceutically acceptable monodentate ligands.

Preferably, the rhenium metal in the compounds of the present inventionis not radioactive.

It was surprisingly found that compounds of formula (I) featuringmonodentate ligands to Re^(I) or Re^(II) do not only demonstrate aerobicstability in solid or dissolved form as previously reported by theinventors (Zobi et al., Inorg. Chem. 2009, 48, 8965-8970), they actuallyrelease carbon monoxide under physiological conditions in a controllablemanner, the rate of release depending on the nature of the monodentateligands and the pH.

Compared to the known [RuCl(glycinato)(CO)₃] complex designated CO-RM-3that is presently investigated for therapeutic carbon monoxide releaseand which has a half-life of about one minute under physiologicalconditions at pH 7.4 and 37° C., rhenium compounds of the invention havebeen demonstrated to provide for a carbon monoxide release with ahalf-life of six minutes to almost an hour. This increased andadjustable delay in the duration and rate of release over CO-RM3 is asignificant advantage for the dosing, timing and targeting options ofcarbon monoxide.

The term “pharmaceutically acceptable” as used herein in the context ofthe monodentate ligands of the inventive compounds is meant to excludethose ligands that no longer allow for medical utility of the inventivecompounds because the adverse effects of the particular ligand, i.e.toxicity, at the dose and the mode administered no longer render thecompound medically useful.

Mono- or also called unidentate ligands are those wherein only one atombinds to the central rhenium ion in the compounds of the invention.

For the compounds of the invention Re(II) is preferred because itscomplexes features 17 electrons, hence, are electronically unsaturatedand thus more reactive.

Next to the necessity for pharmaceutically acceptable and monodentateligands no further structural or functional constraints were identifiedfor the ligands of the compounds of the invention except that theyrequire the presence of either at least one heteroatom, preferably N, O,S and/or P, or at least one carbon-carbon double or triple bond forbinding the rhenium metal of the complex. The group of suitable ligandswas experimentally verified to encompass water, carbon monoxide,halogens, heteroaromatic and alcoholic compounds, i.e. virtually anycompound or atom capable of forming a monodentate rhenium I or IIcomplex together with at least two carbon monoxides.

The ligands can be independently selected from neutral, positively ornegatively charged monodentate ligands. In a preferred embodiment thecompounds of the invention are those, wherein at least one, preferablytwo, more preferably three, most preferably all of L1, L2, L3 and L4 areindependently selected from the group consisting of neutral ornegatively charged, preferably neutral monodentate ligands. Non-limitingexamples of suitable positively charged ligands are pyrazinium,pyrimidinium or 4,4′-bipyrimidinium-type ligands, preferablypyrimidinium. Non-limiting examples of suitable neutral ligands areimidazole, pyridine and pyrazine-type ligands, preferably imidazole.Non-limiting examples of suitable negatively charged ligands arecyanide, hydroxide and halogens, preferably hydroxide and halogens, morepreferably bromide. Further examples are described throughout thedescription.

As mentioned before, the monodentate ligands for practicing theinvention can be selected from an extremely broad repertoire of chemicalcompounds of highly diverse nature, e.g. size, charge, spatial andfunctional arrangement of atoms. Because the present invention is abreakthrough in medicinal rhenium chemistry, vastly expanding theprevious very limited use as radioactive cytotoxin and Tc-modelcompound, there is little information on ligand constraints; and alsothose highly diverse compounds of the invention already tested do notindicate any constraints. Hence, the ligands for practicing theinvention can only be defined functionally without unduly limiting thescope of the invention.

The terms “in each case independently of one another” or “independentlyselected from the group consisting of” in the context of the monodentateligands of the inventive rhenium compounds are meant to indicate thatthe rhenium ligands can be “mixed” ligands, i.e. selected from differentmembers of the group.

The term “independently selected from the group consisting of” is meantto indicate that the four ligands may be selected from the same ordifferent members of the indicated group. It is preferred that at leasttwo ligands are the same; more preferably three ligands are the same andmost preferably all four ligands are the same.

In a more preferred embodiment the compounds of the present inventionare selected from those, wherein at least one, preferably two, morepreferably three, most preferably all of L1, L2, L3 and L4 areindependently selected from the group consisting of:

-   -   alkyl and cycloalkyl comprising at least one heteroatom,        alkenyl, alkynyl, alkylidene, aryl, heteroaryl, arylalkyl,        aryloxy, alkoxy, alkylthio, acyl, alkoxycarbonyl, acyloxy,        acylamino, sulphonylamino, aminosulfonyl, alkylsulfonyl,        carboxy, carboxamide, hydroxyl, oxo, halogen, trifluoromethyl,        nitro, nitrile, isocyanide, alcohol, phosphine, phosphite,        phosphonite, sulphide, sulfoxide and amino or guanidine, each        amino or guanidine optionally mono-, di- or tri-substituted,        preferably mono- or di-substituted by alkyl, acyl or        alkoxycarbonyl, each member of the group optionally substituted        by one to four R″;        wherein each R″ is independently selected from    -   alkyl, alkenyl, alkynyl, alkylidene, cycloalkyl, aryl,        arylalkyl, aryloxy, alkoxy, alkylthio, acyl, alkoxycarbonyl,        acyloxy, acylamino, sulphonylamino, aminosulfonyl,        alkylsulfonyl, carboxy, carboxamide, hydroxy, halogen,        trifluoromethyl, nitro, nitrile and amino optionally mono-, di-        or tri-substituted, preferably mono- or di-substituted by alkyl,        acyl or alkoxycarbonyl,        wherein any members of the group and/or R″ are optionally        halogenated where possible.

Alkyl ligands comprising at least one heteroatom are preferably C₁₋₂₀,more preferably C₁₋₁₂, most preferably C₁₋₆. Cycloalkyl ligandscomprising at least one heteroatom are preferably C₃₋₂₀, more preferablyC₃₋₁₂, more preferably C₃₋₆ and most preferably C₅₋₆. Alkenyl, alkynyland alkylidene ligands are preferably C₂₋₂₀, more preferably C₂₋₁₂, mostpreferably C₂₋₆. Aryl, heteroaryl, arylalkyl and aryloxy ligands arepreferably C₅₋₂₀, more preferably C₅₋₁₂, most preferably C₅₋₆. Alkoxy,alkylthio, acyl, alkoxycarbonyl, acyloxy, acylamino and alkylsulfonylare preferably C₁₋₂₀, more preferably C₁₋₁₂, most preferably C₁₋₆.

With respect to the stereochemistry of the compounds of the invention,it is preferred that the at least two carbon monoxides have a cisconfiguration towards each other, i.e. a cis-[Re^(I/II)(CO)₂L₁₋₄]stereochemistry. In a more preferred embodiment, at least two of thefour ligands L₁₋₄ are of the same type, preferably are halogens, morepreferably bromide and have a trans configuration. This leaves a cisconfiguration for the remaining two ligands resulting in a cis-trans-cisstereochemistry, i.e. cis-trans-cis-[Re^(I/II)(CO)₂(L₁L₄)(L₂L₃)],wherein L₁ and L₄ are preferably halogens, more preferably Br. Hence, ina most preferred embodiment, the invention is directed to cis-trans-cis[Re^(I/II)(CO)₂(halogen)₂(L₂L₃)] compounds, bromides being the preferredhalogens.

Preferred compounds of the invention are those, wherein the at least twocarbon monoxide ligands are in a cis configuration towards each otherand/or at least two of L1, L2, L3 and L4 are halogen, preferably bromideand/or the at least two halogen are in a trans configuration towardseach other.

In a further preferred embodiment the compounds of the present inventionare selected from those, wherein at least one, preferably two, morepreferably three, most preferably four of L1, L2, L3 and L4 areindependently selected from the group consisting of

-   -   C₁₋₁₂ alkyl and C₃₋₁₂ cycloalkyl comprising at least one        heteroatom, preferably C₁₋₆ alkyl and C₃₋₇ cycloalkyl, C₂₋₁₂        alkenyl, preferably C₂₋₆ alkenyl, C₂₋₁₂ alkynyl, preferably C₂₋₆        alkynyl, C₂₋₁₂ alkylidene, preferably C₂₋₆ alkylidene, C₁₋₁₂        alkoxy, preferably C₁₋₆ alkoxy, C₁₋₁₂ alkylthio, preferably C₁₋₆        alkylthio, C₁₋₁₂ acyl, preferably C₁₋₆ acyl, C₂₋₁₂        alkoxycarbonyl, preferably C₂₋₇ alkoxycarbonyl, C₁₋₁₂ acyloxy,        preferably C₁₋₆ acyloxy, C₁₋₁₂ acylamino, preferably C₁₋₆        acylamino, indanyl, indenyl, phenyl naphthyl, heteroaryl        selected from thienyl, furanyl, isoxazolyl, oxazolyl, thiazolyl,        thiadiazolyl, tetrazolyl, pyrazolyl, pyrrolyl, imidazolyl,        pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyranyl,        quinoxalinyl, indolyl, benzimidazolyl, benzoxazolyl,        benzothiazolyl, benzothienyl, quinolinyl, quinazolinyl and        indazolyl and heterocyclyl selected from aziridinyl,        pyrrolidinyl, pyrrolinyl, morpholinyl, thiomorpholinyl,        tetrahydrofuranyl, dioxalanyl, pyranyl, piperidinyl and        piperazinyl, each optionally substituted by one to four R″;        wherein each R″ is independently selected from    -   C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, C₂₋₅ alkylidene, C₃₋₇        cycloalkyl, aryl, arylalkyl, aryloxy, C₁₋₅ alkoxy, C₁₋₅        alkylthio, C₁₋₅ acyl, C₂₋₇ alkoxycarbonyl, C₁₋₅ acyloxy, C₁₋₅        acylamino, sulphonylamino, aminosulfonyl, alkylsulfonyl,        carboxy, carboxamide, hydroxy, halogen, trifluoromethyl, nitro,        nitrile and amino optionally mono-, di- or tri-substituted,        preferably mono- or di-substituted by C₁₋₆ alkyl, C₁₋₆ acyl or        C₂₋₇ alkoxycarbonyl,        wherein any members of the group and/or R″ are optionally        halogenated where possible.

In a further preferred embodiment the compounds of the present inventionare selected from those, wherein at least one, preferably two, morepreferably three, most preferably four of L1, L2, L3 and L4 areindependently selected from the group consisting of

-   (i) halides, preferably Br⁻, OH⁻, CN⁻, ClO₄ ⁻, NO₃ ⁻, NO₂ ⁻, NCO⁻,    NCS⁻, N₃ ⁻ and

-   (ii) water, carbon monoxide and nitrogen, and-   (iii) nitriles, isocyanides, primary, secondary and tertiary amines,    preferably primary and secondary amines, alcohols, phosphines,    phosphates, phosphonites, sulfides, sulfoxides, each optionally    substituted by one to four R″ where possible;    wherein each R and/or R″ is independently selected from    -   alkyl, alkenyl, alkynyl, alkylidene, cycloalkyl, aryl,        arylalkyl, aryloxy, alkoxy, alkylthio, acyl, alkoxycarbonyl,        acyloxy, acylamino, sulphonylamino, aminosulfonyl,        alkylsulfonyl, carboxy, carboxamide, hydroxy, halogen,        trifluoromethyl, nitro, nitrile and amino optionally mono-, di-        or tri-substituted by alkyl, acyl or alkoxycarbonyl, wherein any        of the groups and/or R″ are optionally halogenated where        possible;        preferably, wherein each R and/or R″ is independently selected        from    -   C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₆ alkylidene, C₃₋₇        cycloalkyl, aryl, arylalkyl, aryloxy, C₁₋₅ alkoxy, C₁₋₆        alkylthio, C₁₋₆ acyl, C₂₋₇ alkoxycarbonyl, C₁₋₆ acyloxy, C₁₋₆        acylamino, sulphonylamino, aminosulfonyl, C₁₋₆ alkylsulfonyl,        carboxy, carboxamide, hydroxy, halogen, trifluoromethyl, nitro,        nitrile and amino optionally mono-, di- or tri-substituted,        preferably mono- or di-substituted by C₁₋₆ alkyl, C₁₋₆ acyl or        C₂₋₇ alkoxycarbonyl, wherein any of the groups and/or R″ are        optionally halogenated where possible.

In a further preferred embodiment the compounds of the present inventionare selected from those, wherein at least one, preferably two, morepreferably three, most preferably four of L1, L2, L3 and L4 areindependently selected from the group of 5- or 6-membered heteroatomic(the heteroatoms is/are preferably at least one of N, O, S and/or P)carbon rings, preferably consisting of

wherein X is selected from N, O, S or P with the proviso that forligands with two X, one X may be C, optionally substituted by one tofour R″ where possible;wherein each R″ is independently selected from

-   -   alkyl, alkenyl, alkynyl, alkylidene, cycloalkyl, aryl,        arylalkyl, aryloxy, alkoxy, alkylthio, acyl, alkoxycarbonyl,        acyloxy, acylamino, sulphonylamino, aminosulfonyl,        alkylsulfonyl, carboxy, carboxamide, hydroxy, halogen,        trifluoromethyl, nitro, nitrile and amino optionally mono-, di-        or tri-substituted, preferably mono- or di-substituted by alkyl,        acyl or alkoxycarbonyl, wherein any of the groups and/or R″ are        optionally halogenated where possible;        or preferably, wherein each R″ is independently selected from    -   C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₆ alkylidene, C₃₋₇        cycloalkyl, aryl, arylalkyl, aryloxy, C₁₋₆ alkoxy, C₁₋₆        alkylthio, C₁₋₆ acyl, C₂₋₇ alkoxycarbonyl, C₁₋₆ acyloxy, C₁₋₆        acylamino, sulphonylamino, aminosulfonyl, C₁₋₆ alkylsulfonyl,        carboxy, carboxamide, hydroxy, halogen, trifluoromethyl, nitro,        nitrile and amino optionally mono-, di- or tri-substituted,        preferably mono- or di-substituted by C₁₋₆ alkyl, C₁₋₆ acyl or        C₂₋₇ alkoxycarbonyl, wherein any member of the group and/or R″        are optionally halogenated where possible.

In a further preferred embodiment the compounds of the present inventionare selected from those, wherein at least one, preferably two, morepreferably three, most preferably four of L1, L2, L3 and L4 areindependently selected from the group consisting of 5 to 6-memberedheteroatomic (preferably N, O, S and/or P) carbon rings, preferablythose described directly above, wherein 2 to 8, preferably 2 to 6, morepreferably 2 to 4 of the 5 to 6-membered heteroatomic carbon rings arebound to a common linker, e.g.

wherein X is defined as above and n is preferably 1 to 7, morepreferably 1 to 5; and

-   “linker” denotes a functional group connecting the at least two 5 to    6-membered heteroatomic carbon rings in the ligand,    -   preferably a linker selected from the group consisting of alkyl,        alkenyl, alkynyl, alkylidene, cycloalkyl, aryl, heteroaryl,        arylalkyl, aryloxy, alkoxy, alkylthio, acyl, alkoxycarbonyl,        acyloxy, acylamino, sulphonylamino, aminosulfonyl,        alkylsulfonyl; more preferably a linker selected from the group        consisting of C₁₋₁₂, preferably C₁₋₆ alkyl, C₁₋₁₂, preferably        C₁₋₆ alkenyl, C₁₋₁₂, preferably C₁₋₆ alkynyl, C₁₋₁₂, preferably        C₁₋₆ alkylidene, C₁₋₁₂, preferably C₁₋₆ cycloalkyl, aryl,        heteroaryl, arylalkyl, aryloxy, C₁₋₁₂, preferably C₁₋₆ alkoxy,        C₁₋₁₂, preferably C₁₋₆ alkylthio, C₁₋₁₂, preferably C₁₋₆ acyl,        C₁₋₁₂, preferably C₁₋₆ alkoxycarbonyl, C₁₋₁₂, preferably C₁₋₆        acyloxy, C₁₋₁₂, preferably C₁₋₆ acylamino, sulphonylamino,        aminosulfonyl, C₁₋₁₂, preferably C₁₋₆ alkylsulfonyl;        each compound optionally substituted by one to four R″ where        possible;        wherein each R″ is independently selected from the group        consisting of    -   alkyl, alkenyl, alkynyl, alkylidene, cycloalkyl, aryl,        heteroaryl, arylalkyl, aryloxy, alkoxy, alkylthio, acyl,        alkoxycarbonyl, acyloxy, acylamino, sulphonylamino,        aminosulfonyl, alkylsulfonyl, carboxy, carboxamide, hydroxy,        halogen, trifluoromethyl, nitro, nitrile and amino optionally        mono-, di- or tri-substituted, preferably mono- or disubstituted        by C₁₋₆ alkyl, C₁₋₆ acyl or C₂₋₇ alkoxycarbonyl;-   preferably each R″ is independently selected from the group    consisting of C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, C₂₋₁₂ alkynyl, C₂₋₁₂    alkylidene, C₃₋₁₂ cycloalkyl, aryl, arylalkyl, aryloxy, C₁₋₁₂    alkoxy, C₁₋₁₂ alkylthio, C₁₋₁₂ acyl, C₂₋₁₂ alkoxycarbonyl, C₁₋₁₂    acyloxy, C₁₋₁₂ acylamino, sulphonylamino, aminosulfonyl, C₁₋₁₂    alkylsulfonyl, carboxy, carboxamide, hydroxy, halogen,    trifluoromethyl, nitro, nitrile and amino optionally mono-, di- or    tri-substituted, preferably mono- or disubstituted by C₁₋₆ alkyl,    C₁₋₆ acyl or C₂₋₇alkoxycarbonyl;-   more preferably, wherein each R″ is independently selected from the    group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₆    alkynyl, C₂₋₆ alkylidene, C₃₋₇ cycloalkyl, aryl, arylalkyl, aryloxy,    C₁₋₆ alkoxy, C₁₋₆ alkylthio, C₁₋₆ acyl, C₂₋₇ alkoxycarbonyl, C₁₋₆    acyloxy, C₁₋₆ acylamino, sulphonylamino, aminosulfonyl, C₁₋₆    alkylsulfonyl, carboxy, carboxamide, hydroxy, halogen,    trifluoromethyl, nitro, nitrile and amino optionally mono-, di- or    tri-substituted by C₁₋₅ alkyl, C₁₋₅ acyl or C₂₋₇ alkoxycarbonyl,    wherein any of the linkers and/or R″ are optionally halogenated    where possible;    wherein the rhenium-coordinating atom preferably forms part of an    aromatic system.

In a further preferred embodiment the compounds of the present inventionare selected from those, wherein at least one, preferably two, morepreferably three, most preferably four of L1, L2, L3 and L4 areindependently selected from the group consisting of

wherein n is 1 to 6, preferably 1 to 3;

-   “linker” denotes a functional group connecting the two nitrogen, two    phosphor or two sulphur atoms,    -   preferably a linker selected from the group consisting of alkyl,        alkenyl, alkynyl, alkylidene, cycloalkyl, aryl, heteroaryl,        arylalkyl, aryloxy, alkoxy, alkylthio, acyl, alkoxycarbonyl,        acyloxy, acylamino, sulphonylamino, aminosulfonyl,        alkylsulfonyl; more preferably a linker selected from the group        consisting of C₁₋₁₂, preferably C₁₋₆ alkyl, C₁₋₁₂, preferably        C₁₋₆ alkenyl, C₁₋₁₂, preferably C₁₋₆ alkynyl, C₁₋₁₂, preferably        C₁₋₆ alkylidene, C₁₋₁₂, preferably C₁₋₆ cycloalkyl, aryl,        heteroaryl, arylalkyl, aryloxy, C₁₋₁₂, preferably C₁₋₆ alkoxy,        C₁₋₁₂, preferably C₁₋₆ alkylthio, C₁₋₁₂, preferably C₁₋₆ acyl,        C₁₋₁₂, preferably C₁₋₆ alkoxycarbonyl, C₁₋₁₂, preferably C₁₋₆        acyloxy, C₁₋₁₂, preferably C₁₋₆ acylamino, sulphonylamino,        aminosulfonyl, C₁₋₁₂, preferably C₁₋₆ alkylsulfonyl,        wherein each R is independently selected from the group        consisting of    -   alkyl, alkenyl, alkynyl, alkylidene, cycloalkyl, aryl,        heteroaryl, arylalkyl, aryloxy, alkoxy, alkylthio, acyl,        alkoxycarbonyl, acyloxy, acylamino, sulphonylamino,        aminosulfonyl, alkylsulfonyl, carboxy, carboxamide, hydroxy,        halogen, trifluoromethyl, nitro, nitrile and amino optionally        mono-, di- or tri-substituted, preferably mono- or disubstituted        by C₁₋₆ alkyl, C₁₋₆ acyl or C₂₋₇ alkoxycarbonyl;-   preferably wherein each R is independently selected from the group    consisting of C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, C₂₋₁₂ alkynyl, C₂₋₁₂    alkylidene, C₃₋₁₂ cycloalkyl, aryl, arylalkyl, aryloxy, C₁₋₁₂    alkoxy, C₁₋₁₂ alkylthio, C₁₋₁₂ acyl, C₂₋₁₂ alkoxycarbonyl, C₁₋₁₂    acyloxy, C₁₋₁₂ acylamino, sulphonylamino, aminosulfonyl, C₁₋₁₂    alkylsulfonyl, carboxy, carboxamide, hydroxy, halogen,    trifluoromethyl, nitro, nitrile and amino optionally mono-, di- or    tri-substituted, preferably mono- or disubstituted by C₁₋₆ alkyl,    C₁₋₆ acyl or C₂₋₇ alkoxycarbonyl;-   more preferably, wherein each R is independently selected from the    group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₆    alkynyl, C₂₋₆ alkylidene, C₃₋₇ cycloalkyl, aryl, arylalkyl, aryloxy,    C₁₋₆ alkoxy, C₁₋₆ alkylthio, C₁₋₆ acyl, C₂₋₇ alkoxycarbonyl, C₁₋₆    acyloxy, C₁₋₆ acylamino, sulphonylamino, aminosulfonyl, C₁₋₆    alkylsulfonyl, carboxy, carboxamide, hydroxy, halogen,    trifluoromethyl, nitro, nitrile and amino optionally mono-, di- or    tri-substituted by C₁₋₆ alkyl, C₁₋₆ acyl or C₂₋₇ alkoxycarbonyl,    wherein any of the R are optionally halogenated where possible.

In a further preferred embodiment the compounds of the present inventionare selected from those, wherein at least one, preferably at least two,more preferably at least three and most preferably four of L1, L2, L3and L4 are independently selected from the group consisting ofnucleotides, amino acids, vitamins, preferably vitamin B12, andoligomers of 1 to 10 moieties thereof, preferably

optionally substituted by one to four R″ where possible;wherein each R″ is independently selected from

-   -   alkyl, alkenyl, alkynyl, alkylidene, cycloalkyl, aryl,        heteroaryl, arylalkyl, aryloxy, alkoxy, alkylthio, acyl,        alkoxycarbonyl, acyloxy, acylamino, sulphonylamino,        aminosulfonyl, alkylsulfonyl, carboxy, carboxamide, hydroxy,        halogen, trifluoromethyl, nitro, nitrile and amino optionally        mono-, di- or tri-substituted, preferably mono- or disubstituted        by C₁₋₆ alkyl, C₁₋₆ acyl or C₂₋₇ alkoxycarbonyl;

-   preferably wherein each R″ is independently selected from C₁₋₁₂    alkyl, C₂₋₁₂ alkenyl, C₂₋₁₂ alkynyl, C₂₋₁₂ alkylidene, C₃₋₁₂    cycloalkyl, aryl, arylalkyl, aryloxy, C₁₋₁₂ alkoxy, C₁₋₁₂ alkylthio,    C₁₋₁₂ acyl, C₂₋₁₂ alkoxycarbonyl, C₁₋₁₂ acyloxy, C₁₋₁₂ acylamino,    sulphonylamino, aminosulfonyl, C₁₋₁₂ alkylsulfonyl, carboxy,    carboxamide, hydroxy, halogen, trifluoromethyl, nitro, nitrile and    amino optionally mono-, di- or tri-substituted, preferably mono- or    disubstituted by C₁₋₆ alkyl, C₁₋₆ acyl or C₂₋₇ alkoxycarbonyl;

-   more preferably, wherein each R″ is independently selected from C₁₋₆    alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₆ alkynyl, C₂₋₆ alkylidene,    C₃₋₇ cycloalkyl, aryl, arylalkyl, aryloxy, C₁₋₆ alkoxy, C₁₋₆    alkylthio, C₁₋₆ acyl, C₂₋₇ alkoxycarbonyl, C₁₋₆ acyloxy, C₁₋₆    acylamino, sulphonylamino, aminosulfonyl, C₁₋₆ alkylsulfonyl,    carboxy, carboxamide, hydroxy, halogen, trifluoromethyl, nitro,    nitrile and amino optionally mono-, di- or tri-substituted by C₁₋₅    alkyl, C₁₋₆ acyl or C₂₋₇ alkoxycarbonyl,    wherein any of the R″ are optionally halogenated where possible.

In a most preferred embodiment the compounds of the present inventionhave formula [Re^(II)Br₂(CO)₂(L)₂], preferably cis-trans-cis[Re^(II)Br₂(CO)₂(L)₂], wherein at least one, preferably both L areselected from the group consisting of halides, preferably Br⁻, carbonmonoxide, N-methyl imidazole, benzimidazole, 4-methyl pyridine,imidazole, pyridine, pyridine, C₁₋₆ alkyl cyanide, preferably methylcyanide and alcohol, preferably C₁₋₆ alcohol, more preferably methanolor ethanol, optionally substituted by one to four R″ where possible;

wherein each R″ is independently selected from the group consisting of

-   -   alkyl, alkenyl, alkynyl, alkylidene, cycloalkyl, aryl,        heteroaryl, arylalkyl, aryloxy, alkoxy, alkylthio, acyl,        alkoxycarbonyl, acyloxy, acylamino, sulphonylamino,        aminosulfonyl, alkylsulfonyl, carboxy, carboxamide, hydroxy,        halogen, trifluoromethyl, nitro, nitrile and amino optionally        mono-, di- or tri-substituted, preferably mono- or disubstituted        by C₁₋₆ alkyl, C₁₋₆ acyl or C₂₋₇ alkoxycarbonyl;

-   preferably wherein each R″ is independently selected from the group    consisting of C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl, C₂₋₁₂ alkynyl, C₂₋₁₂    alkylidene, C₃₋₁₂ cycloalkyl, aryl, arylalkyl, aryloxy, C₁₋₁₂    alkoxy, C₁₋₁₂ alkylthio, C₁₋₁₂ acyl, C₂₋₁₂ alkoxycarbonyl, C₁₋₁₂    acyloxy, C₁₋₁₂ acylamino, sulphonylamino, aminosulfonyl, C₁₋₁₂    alkylsulfonyl, carboxy, carboxamide, hydroxy, halogen,    trifluoromethyl, nitro, nitrile and amino optionally mono-, di- or    tri-substituted, preferably mono- or disubstituted by C₁₋₆ alkyl,    C₁₋₆ acyl or C₂₋₇ alkoxycarbonyl;

-   more preferably, wherein each R″ is independently selected from the    group consisting of C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₂₋₆    alkynyl, C₂₋₆ alkylidene, C₃₋₇ cycloalkyl, aryl, arylalkyl, aryloxy,    C₁₋₆ alkoxy, C₁₋₆ alkylthio, C₁₋₆ acyl, C₂₋₇ alkoxycarbonyl, C₁₋₆    acyloxy, C₁₋₆ acylamino, sulphonylamino, aminosulfonyl, C₁₋₆    alkylsulfonyl, carboxy, carboxamide, hydroxy, halogen,    trifluoromethyl, nitro, nitrile and amino optionally mono-, di- or    tri-substituted by C₁₋₆ alkyl, C₁₋₆ acyl or C₂₋₇ alkoxycarbonyl,    wherein any members of the group and/or of the R″ are optionally    halogenated where possible.

Because the monodentate ligands for practicing the invention can beselected from an extremely broad repertoire of chemical compounds ofhighly diverse nature, e.g. size, charge, spatial and functionalarrangement of atoms, the compounds of the invention may comprise atleast one, preferably two diagnostic and/or physiologically activeligands, preferably selected from the group consisting of targetingligands, diagnostic ligands and physiologically, preferably medicallyactive ligands. Hence, the compounds of the invention can function as a“carrier” for “functional” ligands.

The invention includes the use of any compounds described abovecontaining one or more asymmetric carbon atoms which may occur asracemates and racemic mixtures, single enantiomers, diastereomericmixtures and individual diastereomers. All such isomeric forms of thesecompounds are expressly included in the present invention. Eachstereogenic carbon may be in the R or S configuration, or a combinationof configurations. Some of the compounds of the invention can exist inmore than one tautomeric form. The invention includes all suchtautomers.

For all compounds disclosed herein, in the event that the nomenclatureconflicts with the structure, it shall be understood that the compoundis defined by the structure.

All terms as used in this specification, unless otherwise stated, shallbe understood by their ordinary meaning in the art.

The term “heteroatom”, as used herein, for example in the context of theterm heteroaryl shall be understood to mean atoms other than carbon andhydrogen such as and preferably O, N, S and P.

The terms alkyl, alkenyl, alkynyl, alkylidene, etc. shall be understoodas encompassing linear and branched, substituted or non-substitutedforms of carbon-containing chains where structurally possible. In thesecarbon chains one or more carbon atoms can be optionally replaced byheteroatoms, preferably by O, S or N. If N is not substituted, it is NH.The heteroatoms may replace either terminal or internal carbon atomswithin a linear or branched carbon chain. Such groups can be substitutedas herein described by groups such as oxo to result in definitions suchas but not limited to alkoxycarbonyl, acryl, amido and thioxo.

The term “carbocycle” shall be understood to mean a cyclic hydrocarboncontaining from 3 to 20, preferably 3 to 12 carbon atoms, morepreferably 5 or 6 carbon atoms. Carbocycles include hydrocarbon ringscontaining from 3 to 20, preferably 3 to 12 carbon atoms. Thesecarbocycles may be either aromatic or non-aromatic, i.e. cycloalkylsystems or mixed cycloalkyl-aromatic systems. The non-aromatic ringsystems may be mono- or polyunsaturated. Preferred carbocycles includebut are not limited to cyclopropyl, cyclobutyl, cyclopentyl,cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptanyl, cycloheptenyl,phenyl, indanyl, indenyl, benzocyclobutanyl, dihydronaphthyl,tetrahydronaphthyl, naphthyl, decahydronaphthyl, benzocycloheptanyl, andbenzocycloheptenyl. Certain terms for cycloalkyl such as cyclobutanyland cyclobutyl shall be used interchangeably.

The term “cycloalkyl” shall be understood to mean aliphatichydrocarbon-containing rings having from 3 to 20, preferably 3 to 12carbon atoms. These non-aromatic ring systems may be mono- orpolyunsaturated, i.e. the term encompasses cycloalkenyl andcycloalkynyl. The cycloalkyl may comprise heteroatoms, preferably O, Sor N, and be substituted or non-substituted. Preferred and non-limitingcycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptanyl, cycloheptenyl,benzocyclobutanyl, benzocycloheptanyl and benzocycloheptenyl.

The term “heterocyclic” refers to a stable non-aromatic, preferably 3 to20-membered, more preferably 3 to 12-membered, most preferably 5 or6-membered, monocyclic or multicyclic, preferably 8 to 12-memberedbicyclic, heteroatom-containing cyclic compound, that may be eithersaturated or unsaturated. Each heterocycle consists of carbon atoms andone or more, preferably 1 to 4 heteroatoms chosen from nitrogen, oxygen,sulphur and phosphorus. The heterocyclic residue may be bound to theremaining structure of the complete molecule by any atom of the cycle,which results in a stable structure. Exemplary heterocycles include, butare not limited to, pyrrolidinyl, pyrrolinyl, morpholinyl,thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone,dioxalanyl, piperidinyl, piperazinyl, tetrahydrofuranyl,1-oxo-A4-thiomorpholinyl,13-oxa-11-aza-tricyclo[7.3.1.0-2,7]tridecy-2,4,6-triene,tetrahydropyranyl, 2-oxo-2H-pyranyl, tetrahydrofuranyl, 1,3-dioxolanone,1,3-dioxanone, 1,4-dioxanyl, 8-oxa-3-aza-bicyclo[3.2.1]octanyl,2-oxa-5-aza-bicyclo[2.2.1]heptanyl, 2-thia-5-aza-bicyclo[2.2.1]heptanyl,piperidinonyl, tetrahydropyrimidonyl, pentamethylene sulphide,pentamethylene sulfoxide, pentamethylene sulfone, tetramethylenesulphide, tetramethylene sulfoxide and tetramethylene sulfone.

The term “aryl” as used herein shall be understood to mean an aromaticcarbocycle or heteroaryl as defined herein. Each aryl or heteroarylunless otherwise specified includes its partially or fully hydrogenatedderivatives. For example, quinolinyl may include decahydroquinolinyl andtetrahydroquinolinyl; naphthyl may include its hydrogenated derivativessuch as tetrahydronaphthyl. Other partially or fully hydrogenatedderivatives of the aryl and heteroaryl compounds described herein willbe apparent to one of ordinary skill in the art. Naturally, the termencompasses aralkyl and alkylaryl, both of which are preferredembodiments for practicing the compounds of the present invention. Forexample, the term aryl encompasses phenyl, indanyl, indenyl,dihydronaphthyl, tetrahydronaphthyl, naphthyl and decahydronaphthyl.

The term “heteroaryl” shall be understood to mean an aromatic C₃-C₂₀,preferably 5 to 8-membered monocyclic or preferably 8 to 12-memberedbicyclic ring containing 1 to 4 heteroatoms such as N, O, P and S.Exemplary heteroaryls comprise aziridinyl, thienyl, furanyl, isoxazolyl,oxazolyl, thiazolyl, thiadiazolyl, tetrazolyl, pyrazolyl, pyrrolyl,imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyranyl,quinoxalinyl, indolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl,benzothienyl, quinolinyl, quinazolinyl, naphthyridinyl, indazolyl,triazolyl, pyrazolo[3,4-b]pyrimidinyl, purinyl, pyrrolo[2,3-b]pyridinyl,pyrazole[3,4-b]pyridinyl, tubercidinyl, oxazo[4,5-b]pyridinyl andimidazo[4,5-b]pyridinyl.

Terms which are analogues of the above cyclic moieties such as aryloxyor heteroaryl amine shall be understood to mean an aryl, heteroaryl,heterocycle as defined above attached to its respective group.

As used herein, the terms “nitrogen”, “sulphur” and phosphorus includeany oxidized form of nitrogen, sulphur and phosphorus and thequaternized form of any basic nitrogen as long as the resulting compoundis chemically stable. For example, an —S—C₁₋₆ alkyl radical shall beunderstood to include —S(O)—C₁₋₆alkyl and —S(O)₂—C₁₋₆alkyl.

The compounds of the invention are only those which are contemplated tobe ‘chemically stable’ as will be appreciated by those skilled in theart. For example, compounds having a ‘dangling valency’ or a ‘carbanion’are not compounds contemplated by the inventive concept disclosedherein.

Pharmaceutically acceptable salts of the compounds of the inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases, preferably non-oxidizing inorganic and organicacids and bases. Examples of suitable acids include hydrochloric,hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfuric, tartaric,acetic, citric, ethanesulfonic, formic, benzoic, malonic,naphthalene-2-sulfuric and benzenesulfonic acids. Salts derived fromappropriate bases include alkali metal (e.g., sodium), alkaline earthmetal (e.g., magnesium), ammonium and N—(C₁₄ alkyl)₄ ⁺ salts.

In another aspect, the present invention relates to a pharmaceuticalcomposition comprising a pharmaceutically effective amount of at leastone compound according to the invention as described above andoptionally one or more pharmaceutically acceptable carriers and/oradjuvants.

The pharmaceutical compositions of the present invention typicallycomprise a pharmaceutically acceptable excipient, carrier, buffer,stabiliser, preservative and/or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere unduly with the efficacy of the active ingredient. The precisenature of the carrier or other material may depend on the route ofadministration, e.g. oral, intravenous, subcutaneous, nasal,intramuscular, intraperitoneal or suppository routes. Pharmaceuticalcompositions for oral administration may be in tablet, capsule, powderor liquid form. A tablet may include a solid carrier such as gelatin oran adjuvant or a slow-release polymer. Liquid pharmaceuticalcompositions generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included. Pharmaceutically acceptable amounts of other solventsmay also be included, in particular where they are required fordissolving the particular metal carbonyl compound contained in thecomposition. For intravenous, cutaneous or subcutaneous injection orinjection at the site of affliction the active ingredient will typicallybe in the form of a parenterally acceptable solution which ispyrogen-free and has suitable pH, isotonicity and stability. Those ofskill in the pharmaceutical art are capable of preparing suitablesolutions using, for example, isotonic vehicles such as physiologicalsaline, Ringers injection solution and Ringer's lactate solution forinjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required. Delivery systems forneedle-free injection are also known, and compositions of the inventionfor use with such systems may be prepared accordingly.

In a further aspect, the present invention relates to a use of acompound of the invention as defined above for the preparation of amedicament for the treatment and/or protection of patients having orbeing prone to produce a disease or medical condition responsive tocarbon monoxide treatment, preferably a disease or medical conditioninvolving hypoxic, anoxic and/or inflamed mammalian tissue, preferably atissue selected from the group consisting of heart, lung, liver, brain,gut, kidney, muscle, bone, skin and eye, preferably a disease or medicalcondition selected from the group consisting of cardiovascular diseases,preferably cardiac hypoxia, cardiac infarction, cardiac hypertrophy,arteriosclerosis and hypertension; ischemia-reperfusion injury,inflammatory diseases, preferably asthma or angina; traumatic injury,preferably of the brain, kidney or liver; transplant rejection,preferably allograft and xenograft rejection, platelet aggregationand/or monocyte activation; neuron degeneration of the nervous system,radiation damage, cancer, penile erectile dysfunction, adult respiratorydistress syndrome, and disorders of the circadian rhythm of mammals,preferably jet lag.

The above diseases or medical conditions have been established as beingresponsive to carbon monoxide administration (see WO 02/092075 andJohnson et al., Metal carbonyls in Medicine, Angew. Chem. Int. Ed. 2003,42, 3722-3729.)

The pharmaceutical composition/medicament is for administration priorto, after or concomitantly to a medical condition.

As used herein, a “patient” means any mammal that may benefit from atreatment with the compounds of the invention. Preferably, a “patient”is selected from the group consisting of laboratory animals (e.g. mouseor rat), domestic animals (including e.g. guinea pig, rabbit, pig,sheep, goat, camel, cow, horse, donkey, cat, or dog), or primatesincluding human beings. It is particularly preferred that the “patient”is a human being.

As used herein, “treat”, “treating” or “treatment” of a disease, medicalcondition or disorder means accomplishing one or more of the following:(a) reducing the severity of the disorder; (b) limiting or preventingdevelopment of symptoms characteristic of the disorder(s) being treated;(c) inhibiting worsening of symptoms characteristic of the disorder(s)being treated; (d) limiting or preventing recurrence of the disorder(s)in patients that have previously had the disorder(s); and (e) limitingor preventing recurrence of symptoms in patients that were previouslysymptomatic for the disorder(s).

As used herein, “administering” includes in vivo administration, as wellas administration directly to tissue ex vivo, such as vein grafts.Administration may be effected for the prevention, i.e. before clinicaloccurrence of a disease or disorder, or for treatment, i.e. afterclinical occurrence of a disease or disorder.

In a preferred embodiment, the present invention is directed to the useof the above-mentioned compounds for preparing a medicament for thetreatment of a disease or medical condition selected from the groupconsisting of selected from the group consisting of cardiovasculardiseases, preferably cardiac hypoxia, cardiac infarction, cardiachypertrophy, arteriosclerosis and hypertension; ischemia-reperfusioninjury, inflammatory diseases, preferably asthma or angina; traumaticinjury, preferably of the brain, kidney or liver; transplant rejection,preferably allograft and xenograft rejection, platelet aggregationand/or monocyte activation; neuron degeneration of the nervous system,radiation damage, cancer, penile erectile dysfunction, adult respiratorydistress syndrome, and disorders of the circadian rhythm of mammals,preferably jet lag.

Methods of Use

In a further aspect, the present invention relates to a method oftreating and/or protecting patients having or being prone to develop adisease or medical condition responsive to carbon monoxide treatment asdiscussed above, the method comprising the administration of atherapeutically effective amount of at least one compound of theinvention as defined above or a prodrug thereof or an effective amountof the pharmaceutical composition of the invention as defined above to apatient in need thereof.

An “effective amount” is an amount of a therapeutic agent sufficient toachieve the intended purpose. The effective amount of a giventherapeutic agent will vary with factors such as the nature of theagent, the route of administration, the size and species of the animalto receive the therapeutic agent, and the purpose of the administration.The effective amount in each individual case may be determinedempirically by a skilled artisan according to established methods in theart.

For therapeutic or prophylactic use the compounds of the invention maybe administered in any conventional dosage form in any conventionalmanner. Routes of administration include, but are not limited to,intravenously, intramuscularly, cutaneously, subcutaneously,intrasynovially, by infusion, sublingually, transdermally, orally, ortopically. The preferred modes of administration is intravenous.

The compounds may be administered alone or in combination with adjuvantsthat enhance stability of the compounds, facilitate administration ofpharmaceutical compositions containing them in certain embodiments,provide increased dissolution or dispersion, provide adjunct therapy,and the like, including other active ingredients. Advantageously suchcombination therapies utilize lower dosages of the conventionaltherapeutics, thus avoiding possible toxicity and adverse side-effectsincurred when those agents are used as monotherapies. The abovedescribed compounds may be physically combined with the conventionaltherapeutics or other adjuvants into a single pharmaceuticalcomposition. Reference is this regard may be made to Cappola et al.:U.S. patent application Ser. No. 09/902,822, PCT/US 01/21860 and U.S.provisional application No. 60/313,527, each incorporated by referenceherein in their entirety. Advantageously, the compounds may then beadministered together in a single dosage form. In some embodiments, thepharmaceutical compositions comprising such combinations of compoundscontain at least about 5%, but more preferably at least about 20%, of acompound of formula (I) (w/w) or a combination thereof. The optimumpercentage (w/w) of a compound of the invention may vary and is withinthe purview of those skilled in the art. Alternatively, the compoundsmay be administered separately (either serially or in parallel).Separate dosing allows for greater flexibility in the dosing regimen.

As mentioned above, dosage forms of the compounds described hereininclude pharmaceutically acceptable carriers and adjuvants known tothose of ordinary skill in the art. These carriers and adjuvantsinclude, for example, ion exchangers, alumina, aluminium stearate,lecithin, serum proteins, buffer substances, water, salts orelectrolytes and cellulose-based substances. Preferred dosage formsinclude, tablet, capsule, caplet, liquid, solution, suspension,emulsion, lozenges, syrup, reconstitutable powder, granule, suppositoryand transdermal patch. Controlled release dosage forms with or withoutimmediate release portions are also envisaged. Methods for preparingsuch dosage forms are known (see, for example, H. C. Ansel and N. G.Popovish, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5^(th)ed., Lea and Febiger (1990)). Dosage levels and requirements arewell-recognized in the art and may be selected by those of ordinaryskill in the art from available methods and techniques suitable for aparticular patient. In some embodiments, dosage levels range from about0.1-100 g/dose for a 70 kg patient. Although one dose per day may besufficient, up to 5 doses per day may be given. For oral doses, up to2000 mg/day or more may be required.

Reference in this regard may also be made to U.S. provisionalapplication No. 60/339,249. As the skilled artisan will appreciate,lower or higher doses may be required depending on particular factors.For instance, specific doses and treatment regimens will depend onfactors such as the patient's general health profile, the severity andcourse of the patient's disorder or disposition thereto, and thejudgment of the treating physician.

For further information on how to formulate and administer the carbonmonoxide releasing rhenium compounds of the invention, reference is madeto the following documents WO 2005/013691 A1, WO 2009/013612, US2007/0207993, US 2007/0207217, US 2006/0233890, US 2006/0148900 and US2004/0067261.

In another aspect, the present invention relates to compounds of formula(I) as described above, preferably non-radioactive compounds, morepreferably with the proviso that the compound is not selected from thegroup explicitly disclosed in the recent publication of the presentinventors (Zobi et al., Inorg. Chem. 2009, 48, 8965-8970), the grouppreferably consisting of [Re^(II)Br₄(CO)₂]²⁻,[Re^(II)Br₂(CO)₂(pyridine)₂], [Re^(II)Br₂(CO)₂(imidazole)₂],[Re^(II)(CO)₂(HOCH₃)₄]²⁺, [Re^(II)Br₃(CO)₂(N≡CCH₃)]⁻,[Re^(I)Br₃(CO)₃]²⁻, [Re^(I)Br₂(CO)₂(imidazole)₂]⁻,[Re′Br₂(CO)₂(N≡CCH₃)₂] and (Re^(I)(CO)₃(H₂O)₃]⁺.

The following tables, figures and examples are merely illustrative ofthe present invention and should not be construed to limit the scope ofthe invention as indicated by the appended claims in any way.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 graphically represents a typical absorption spectrum of theconversion of deoxy-myoglobin (Mb) to carbon monoxide myoglobin (MbCO)by a cis-trans-[Re^(II)(CO)₂Br₂L₂]n complex exemplified with a 30 μM Mbsolution after the addition of compound 4 (30 μM, 25° C., 0.1 Mphosphate buffer, pH 7.4).

FIGS. 2 to 7 graphically represent the results of the pH-dependent rateof CO release of different cis-trans-[Re^(II)(CO)₂Br₂L₂]^(n) complexesunder conditions of 30 μM Mb (myoglobin), 25° C., 0.1 M phosphatebuffer, pH 7.4 (), 6.3 (▴), 5.8 (▪).

FIG. 8. Cytoprotective effects of conjugates B₁₂-CORM-2 and B₁₂CO-RM-4(30 μM) against “ischemia-reperfusion” injury (I/R). Cell damage inneonatal rat ventricular cardiomyocytes (NRCs) after 16 h of ischemia(hypoxia, aglycemia, and acidosis). CO-RMs or the aliquot of solvent(DMSO) were added to the cell culture medium at the onset of reperfusionperiod of 9 h at 30 μM concentration. Bars indicate the % of PI-positive(dead) cells. ** denotes p<0.01 when I/R B₁₂-CORM-2-treated cells arecompared to I/R control. # denotes p<0.05 when normoxic cells arecompared with I/R control.

FIGS. 9A-9B. Regulation of oxygen availability and the intracellularreduced glutathione content by Re-CORMs. A: Reduction in oxygen contentin the incubation medium within 10 min after addition of 60 μM of 2, 6and B₁₂-CORM-2; N=6-8. Given are means±SEM. B: Intracellular reducedglutathione (GSH) content in NRCs exposed to 16 h of ischemia and 10 minof reperfusion in the presence or absence of CO-RMs. N=4 Data aremeans±SEM.

EXAMPLES Example 1 Methods of Preparation

The compounds of the present invention can be prepared without any undueburden or inventive skill by any appropriate conventional syntheticstrategy known to those of skill in organic and inorganic chemistry, inparticular in view of the recent disclosure of the present inventors inInorg. Chem. 2009, 48, 8965-8970, the synthetic routes of which areincorporated in toto by reference herewith.

Chemicals and solvents were purchased from standard sources, distilledand/or degassed where necessary prior to use. (NEt₄)₂[ReBr₃(CO)₃],compounds 1, 2, 6, 7, 12 and 13 were synthesized as previously described(Zobi et al., Inorg. Chem. 2009, 48, 8965-8970; Alberto et al. J. Chem.Soc. Dalton Trans. 1994, 2815-2820; Abram et al., Z. Anorg. Allg. Chem.1996, 622, 813-818). All other complexes were synthesized under nitrogenwith standard techniques. Elemental analyses (EA) were performed on aLeco CHNS-932 elemental analyser. IR spectra were recorded in aPerkinElmer Spectrum BX FT-IR spectrometer. Crystallographic data werecollected at 183(2) K with Mo K_(α) radiation (λ=0.7107 Å) that wasmonochromated with help of a graphite on an Oxford Diffraction Xcalibursystem with a Ruby detector. Suitable crystals were covered with oil(Infineum V8512), mounted on top of a glass fiber and immediatelytransferred to the diffractometer. The program suite CrysAlis^(Pro) wasused for data collection, semi-empirical absorption correction and datareduction. Structures were solved with direct methods using SIR97 andwere refined by full-matrix least-squares methods on F² with SHELXL-97.The structures were checked for higher symmetry using the programPlaton.

Example 2 Detection of CO Release Using the Myoglobin Assay

The release of CO from the compounds of the invention was assessedspectrophotometrically by measuring the conversion of deoxymyoglobin(Mb) to carbonmonoxy myoglobin (MbCO) as previously reported (Clark etal., Circ. Res. 2003, 93, e2-8; Motterlini et al., Circ. Res. 2002, 90,E17-24; Motterlini et al., Curr. Pharm. Des. 2003, 9, 2525-2539). Asmall aliquot of a freshly prepared concentrated solution of theselected Re complex (compound 2 in methanol, in DMSO for all othercomplexes) was added to 1 ml of the Mb solution in phosphate bufferprepared at different pHs (7.4, 6.3 and 5.8; final concentrations: 30 μMfor Re complex and Mb). Changes in the Mb spectra were recorded overtime at 25° C. The methanol or DMSO content of the solution neverexceeded 0.5%. The amount of MbCO formed was determined by measuring theabsorbance at 540 nm (extinction coefficient=15.4 M-1 cm-1). The MbCOconcentration was plotted over time and directly related to theequivalents of CO released from the compounds. The half-life of COrelease from the Re compounds at different pH's was then estimated fromthe graphs. Control experiments were run under identical conditions butwithout addition of the metal complexes. All manipulations wereperformed under a pure N₂ atmosphere in a wet box.

Synthesis of [ReBr₂(CO)₂(MeIm)₂] (3).

100 mg of 2 (0.122 mmol) were suspended in 15 ml of DME and 32 mg ofN-methylimidazole (MeIm, 3 eq.) were added. The mixture was heated to60° C. for 3.5 h and stopped when the red suspension had become a yellowsolution and a yellow precipitate had formed. The mixture was filteredwhile still hot. A bright yellow solid of 3 was collected, dried invacuo and recrystallized from a CH₂Cl₂/hexane mixture giving dark redcrystals. Yield: 49 mg, 71%. Anal. Calc. for C₁₀H₁₂Br₂N₄C₂Re (566.2): C,21.21%; H, 2.14%; N, 9.89%. Found: C, 21.89%; H, 2.32%; N, 9.64%. I.r.(solid state, KBr, cm⁻¹): ν_(C≡O) 1982, 1825. Single crystals suitablefor x-ray diffraction were grown by slow diffusion of hexane into aCH₂Cl₂ solution of the compound.

Synthesis of [ReBr₂(CO)₂(BzIm)₂] (4).

100 mg of 2 (0.122 mmol) were suspended in 15 ml of DME and 46 mg ofbenzimidazole (BzIm, 3 eq.) were added. The mixture was heated to 60° C.for 3.5 h and stopped when the red suspension had become a yellowsolution and a yellow precipitate had formed. The mixture was filteredwhile still hot. A bright yellow solid of 4 was collected and dried invacuo. The crude product was purified by loading a CH₃OH solution of 4onto a chromatofix C18 filter. This was washed with a 15% CH₃OH solutionin water and then extracted with CH₃OH. Yield: 39 mg, 50%. Anal. Calc.for C₁₆H₁₂Br₂N₄C₂Re (638.3): C, 30.11%; H, 1.89%; N, 8.78%. Found: C,29.99%; H, 1.82%; N, 8.47%. I.r. (solid state, KBr, cm⁻¹): ν_(C≡O) 1992,1833. Single crystals suitable for x-ray diffraction were grown by slowdiffusion of hexane into a CH₂Cl₂ solution of the compound giving darkred crystals.

Synthesis of [ReBr₂(CO)₂(4-pic)₂] (5).

100 mg of 2 (0.122 mmol) were suspended in 10 ml of DME and ca. 10 eq.4-methylpyridine (4-pic, 100 μl) were added. After stirring overnight, abrownish precipitate was filtered off and dried in vacuo. The crudeproduct was recrystallized from a CH₂Cl₂/hexane mixture giving dark redcrystals of 5 which were found suitable for x-ray diffraction. Yield: 50mg, 70%. Anal. Calc. for C₁₄H₁₄Br₂N₂C₂Re (588.3): C, 28.58%; H, 2.40%;N, 4.76%. Found: C, 28.39%; H, 2.69%; N, 4.88%. I.r. (solid state, KBr,cm⁻¹): ν_(C≡O) 1992, 1830.

Synthesis of [Et₄N][ReBr₂(CO)₂(4-picoline amine)₂] (15).

100 mg of 2 (0.122 mmol) were suspended in 15 ml of DME and 39 mg of4-picoline amine (3 eq.) were added. The mixture was stirred at roomtemperature for 48 h. A brown precipitate formed the was filtered. Abrown solid of 15 was collected and dried in vacuo. Yield: 50 mg, 55%.¹H-NMR spectrum (in DMSO-d6): 8.64 (doublet, 2H), 7.47 (doublet, 2H),4.08 (singlet, 2H). CV (in DMF, 0.1M TBAPF₆ as electrolyte):Re^(I)-Re^(II) couple at 0V vs Ag/AgCl. Fully reversible. I.r. (solidstate, KBr, cm⁻¹): ν_(C≡O) 1875, 1776.

Synthesis of ReBr₂(CO)₂(4,4′-bipy)] (16)

50 mg of 2 (0.061 mmol) were suspended in 5 ml of DME and 20 mg of4,4′-bipyridine (4,4′-bipy, 0.12 mmol, 2 eq.) were added. The suspensionwas heated to 60° C. for 3.5 h and stopped when the red suspension hadbecome a deep red solution. The mixture was allowed to cool to roomtemperature and then filtered. The dark red solution was dried in vacuoleaving 16 as a dark red solid. The crude product was recrystallizedfrom a CH₂Cl₂/hexane mixture. This procedure lead to single X-rayquality crystals of 16 and, due to decomposition of the product, a darkprecipitate. Crystals of 16 were separated from the dark powder anddried in vacuo. Yield: 8 mg, 22%. Anal. Calc. for C₂₂H₁₆Br₂N₄O₂Re(714.4): C, 36.99%; H, 2.26%; N, 7.84%. Found: C, 36.90%; H, 2.33%; N,7.49%. I.r. (solid state, KBr, cm⁻¹): ν_(C≡O) 1983, 1830. MS (ESI⁺): m/z715.6, i.e [M+1]⁺, calculated 715.4.

Synthesis of (Et₄N)₂[Re₂Br₆(CO)₄(pyz)] (17)

50 mg of 2 (0.061 mmol) were suspended in 7 ml of DME and 15 mg ofpyrazine (pyz, 0.19 mmol, 3 eq.) were added. The mixture was heated to60° C., allowed to react for 3.5 h and then filtered while still hot. Ahighly electrostatically charged brown solid of 17 was collected anddried in vacuo. Yield: 35 mg, 88%. Anal. Calc. for C₂₄H₄₄Br₆N₄O₄Re₂(1304.5): C, 22.10%; H, 3.40%; N, 4.29%. Found: C, 22.89%; H, 3.36%; N,4.12%. I.r. (solid state, KBr, cm⁻¹): ν_(C≡O) 1984, 1858. MS (ESI⁻): m/z964.1, i.e [M−Br]⁻, calculated 964.06. Single crystals suitable forX-ray diffraction were grown by slow diffusion of hexane into a CH₂Cl₂solution of the compound.

Synthesis of (Et₄N)₂[Re₂Br₆(CO)₄(pym)] (18)

50 mg of 2 (0.061 mmol) were suspended in 7 ml of DME and 15 mg ofpyrimidine (pym, 0.19 mmol, 3 eq.) were added. The mixture was heated to60° C. and allowed to react for 3.5 h. The solution was allowed to coolto room temperature and then filtered. The solid, identified as amixture of 2, 18 and salts was discarded. The solution was dried invacuo giving an orange-brown powder of 18 which was recrystallized froma CH₂Cl₂/hexane mixture giving red-orange needles. Yield: 20 mg, 50%.Anal. Calc. for C₂₄H₄₄Br₆N₄C₄Re₂ (1304.5): C, 22.10%; H, 3.40%; N,4.29%. Found: C, 22.61%; H, 3.73%; N, 4.69%. I.r. (solid state, KBr,cm⁻¹): ν_(C≡O) 1985, 1844. MS (ESI⁻): m/z 964.1, i.e [M−Br]⁻, calculated964.06. Attempts to obtain single crystals suitable for X-raydiffraction always gave twinned red-orange needles of poor quality.

Synthesis of (Et₄N)₂[Re₂Br₆(CO)₄(Br-pym)] (19)

The same procedure for the synthesis of 18 was employed but using5-Br-pyrimidine instead. Yield: 26 mg, 63%. Anal. Calc. forC₂₄H₄₃Br₄C₄Re₂ (1383.4): C, 20.84%; H, 3.13%, N, 4.05%. Found: C,21.16%; H, 3.45%; N, 4.12%. I.r. (solid state, KBr, cm⁻¹): ν_(C≡O) 1984,1854. MS (ESI⁻): m/z 1042.5, i.e [M−Br]⁻, calculated 1043. Attempts toobtain single crystals suitable for X-ray diffraction always gavetwinned red-orange needles of poor quality.

Synthesis of (Et₄N)[ReBr₃(CO)₂(pyd)] (20)

50 mg of 2 (0.061 mmol) and 2.5 mg of pyridazine (pyd, 0.031 mmol, 0.5eq.) were suspended in 5 ml of DME. The mixture was heated to 60° C. andallowed to react for 5 h. The solution was allowed to cool for a fewminutes and then filtered while still warm. The red-orange solid wasidentified as unreacted 2. The solution was dried in vacuo giving anorange-brown powder of 9 which was recrystallized from a CH₂Cl₂/hexanemixture giving yellow plates which were found suitable for X-raydiffraction. Yield: 10 mg, 25%. Anal. Calc. for C₁₄H₂₄Br₃N₃C₂Re (692.3):C, 24.29%; H, 3.49%; N, 6.07%. Found: C, 23.99%; H, 3.52%; N, 6.20%.I.r. (solid state, KBr, cm⁻¹): ν_(C≡O) 1984, 1858. MS (ESI⁻): m/z 562.8,i.e [M]⁻, calculated 562.0.

Synthesis of B₁₂-CORM-2.

Cyanocobalamin (B₁₂, 10 mg, 7.4 μmol) and 2 (10 mg, 12 μmol) were addedas solids to round bottom flask. Methanol (6 mL) was added, stirringbegan and then the flask was lowered into an oil bath preheated to 50°C. The temperature was then lowered to 40° C. within 1 h. After 1.5 hHPLC analysis showed a single B₁₂ derivative. Heating was stopped andthe solvent removed under reduced pressure. The resulting red powder waswashed several times with CH₂Cl₂ (ca. 1.7 eq. of [Et4N]Br was thusrecovered) and then acetone until washings were clear. CompoundB₁₂-CORM-2 was thus obtained as a red microcrystalline powder. Yield:12.8 mg, 98%. HPLC analysis showed a single peak with a retention timeof 22 min. Analytical data for B₁₂-CORM-2: ESI-MS analysis (positivemode) gave peaks at 1758.4 m/z [M+H⁺]⁺ and 879.8 m/z [M+H⁺]²⁺. I.r.(solid state, KBr, cm⁻¹): ν_(C≡N) 2184,

_(C≡O) 1989, 1839.

Synthesis of B₁₂-CORM-4.

Cyanocobalamin (400 mg, 0.3 mmol) was dissolved in 10 mL of anhydrousDMSO and carbonyldiimidazole (CDI, 1 g, 6.2 mmol) was added. Thesolution was stirred and heated to 60° C. for 12 h. The solution wasallowed to cool to room temperature (RT) and then slowly added to arapidly stirring mixture of 300 mL 1:1 diethyl ether:chloroform. The redprecipitate was collected by vacuum filtration, washed with 50 mL ofacetone and vacuum dried. Yield: 395 mg. This product was not purifiedbut used directly for the next reaction. 100 mg of the activated vitaminwas dissolved in 5 mL of anhydrous DMSO and 4-picolylamine (100 μl, 1mmol) was added. The solution was stirred at RT for 30 min and thenslowly added to a rapidly stirring mixture of 150 mL 1:1 diethylether:chloroform. The red precipitate was collected by vacuumfiltration, washed with 30 mL of acetone and vacuum dried. Yield: 109mg. This precipitate consists of a mixture of products. Thecyanocobalamin derivative with HPLC retention time of 15 min waspurified by HPLC. Yield 45 mg. This derivative was reacted with 2 asdescribed for B₁₂-CORM-2. Compound B₁₂-CORM-4 was obtained as a redmicrocrystalline powder. Yield: 4.9 mg, 95. HPLC analysis showed asingle peak with a retention time of 25 min. Analytical data forB₁₂-CORM-4: ESI-MS analysis (positive mode) gave peaks at 1893.4 m/z[M+H⁺]⁺ and 946.8 m/z [M+H⁺]²⁺. I.r. (solid state, KBr, cm⁻¹): ν_(C≡N)2184,

_(C≡O) 1989, 1839.

Synthesis of Complexes.

A summary of the reactions of compound 2 with different mono-, bi- andtridentate ligands is given in the graphic schemes below. As previouslyshown, complex 2 reacts well with monodentate nitrogen containingaromatic ligands (Zobiet al., Inorg. Chem. 2009, 48, 8965-8970). Thus,the reaction with N-methylimidazole (MeIm), 4-picoline (4-pic) orbenzimidazole (BzIm) gave the correspondingcis-trans-[Re^(II)(CO)₂Br₂L₂] complex (compound 3 with L=MeIm, compound4 with L=BzIm, compound 5 with L=4-pic). The direct substitutionreaction of 2 with this type of ligands allowed to isolate 3, 4 and 5 ina short time and in good isolated yields (>50%). With the exception of3, which was found to be hydroscopic and slowly decomposed over time, 4and 5 appear indefinitely stable as solids under aerobic conditions.

The cyanocobalamin (B₁₂) derivatives of 17-electrons rhenium dicarbonylspecies shown in Scheme 3 are a second generation of Re^(II)-basedCO-RMs. They show several improved features over the original family.The derivatives: a) are fully water soluble and biocompatible, b) showimproved stability in aqueous aerobic media over the metal complexalone, c) are non-toxic towards cultured cardiomyocytes, d) protectthese cells against get injury and e) after CO release, in water underaerobic conditions, the rhenium complex is oxidized to ReO₄ ⁻ which isamong the least toxic of all of the rare inorganic compounds.

Complexes 3-5 are soluble in common organic solvents like methanol,CH₂Cl₂, acetonitrile or DMSO and even under aerobic conditions arestable for days. X-ray quality crystals of 3, 4 and 5 could be grownfrom CH₂Cl₂. In all cases the ligands L substituted the two bromidestrans to the CO's resulting in [Re^(II)(CO)₂Br₂L₂] complexes with acis-trans-cis arrangement of the ligands. Since the cis arrangement ofthe two COs is the only structural compulsion, other isomers areexpected as well. A common feature of the compounds 3-5 is the bendingof the two trans bromides away from the CO's with an average Br—Re—Brangle of 171.5°. Bond lengths and angles of 3 and 5 were found similarto the related imidazole and pyridine complexescis-trans-[Re^(II)(CO)₂Br₂(Im)₂] (6) andcis-trans-[Re^(II)(CO)₂Br₂(py)₂] (7) previously described (Zobiet al.,Inorg. Chem. 2009, 48, 8965-8970.). When reacted with othersheterocyclic aromatic ligands like THF or thiophene, 2 was alwaysrecovered unreacted indicating that, under similar experimentalconditions, these ligands are not strong enough to replace the boundbromides even if present in large excess.

While the MeIm and 4-pic ligands were mainly selected in order toprovide data for comparison to the Im and py adducts 6 and 7, BzIm wasalso selected as a simple model for the interaction of 2 with purinebases. In 4 the two BzIm ligands are found in a head-to-tail (HT)conformation. Our previous results of the interaction of DNA bases withthe fac-[Re^(I)(CO)₃(H₂O)₃]⁺ complex have shown that neither hydrogenbonding interactions nor steric factors are important in determining theorientation of the bases around the Re′ core. The bases were found to beable to freely rotate around the metal center and we have shown that thedifferent head-to-head (HH) or HT conformers observed in the solid statestructures of the adducts are a result of packing effects (Zobi et al.,Inorg. Chem. 2004, 43, 2087-2096). The BzIm and other purine-typeligands are expected to be able to freely rotate around thecis-[Re^(II)(CO)₂]²⁺ core. Thus, the HT conformer observed in the solidstate structure of 4 is unlikely to be the predominant form in solution.

Reaction of 2 with bidentate bipyridine or phenantroline type ligandsproceeded smoothly with no reduction of the Re^(II) center. Thus,4,4′-dimethyl-2,2′-bipyridine (4,4′-Mebipy),1,10-phenantroline-5,6-dione (phd), 4,7-dimethyl-1,10-phenantroline(4,7-Mephen) and 2,2′-dipyridylamine (2,2′-dipy-NH) gave thecorresponding cis-trans-[Re^(II)(CO)₂Br₂N∩N] complexes (8 withN∩N=4,4′-Mebipy, 9 with N∩N=phd, 10 with N∩N=4,7-Mephen and 11 withN∩N=2,2′-dipy-NH; see scheme 2).

TABLE 1 Spectroscopic and electrochemical properties of complexes 2-14.E½, λ_(max) Complex v_(CO) (cm⁻¹)^(a) (mV)^(b) (nm)^(c) [Re(CO)₂Br₄]²⁻(2)¹ 1972, 1796 −120 412 [Re(CO)₂Br₂(Melm)₂] (3) 1982, 1825 dec. 418[Re(CO)₂Br₂(Bzlm)₂] (4) 1992, 1833 −195 418 [Re(CO)₂Br₂(4-pic)₂] (5)1992, 1830 −90 423 [Re(CO)₂Br₂(lm)₂] (6)¹ 1988, 1826 dec. 418[Re(CO)₂Br₂(py)₂] (7)¹ 1990, 1825 −76 425 [Et₄N][Re(CO)₂Br₂(4- 1875,1776 0 picolineamine)₂] (15) ^(a)KBr. ^(b)Potentials are reported vs.Ag/AgCl reference electrode in CH₃OH for 2-7 and with 0.1M TBAPF₆ as anelectrolyte. All processes are one electron and refer to theRe^(II → Re) ^(I) reduction for 2-7 and to Re^(I) → Re^(II) oxidationfor 15 in DMF and in CH₂Cl₂ for all others.

TABLE 2 Crystallographic data for compounds 3-5. Comp. 3 4 5 FormulaC₁₀H₁₂Br₂—N₄O₂Re C₁₆H₁₂Br₂—N₄O₂Re C₁₄H₁₄Br₂—N₂O₂Re FW 566.26 638.32588.29 T, K    183(2)    183(2)    183(2) space group C2/c C2/c Pccacrystal system monoclinic monoclinic orthorhombic Z 8 4 4 a, Å 30.1515(16) 10.6383(4) 13.33059(15) b, Å  7.62680(19) 12.4396(3)8.38345(9) c, Å 15.1080(7) 13.7647(4) 15.52929(19) β, deg 119.758(7) 99.358(3) 90 V, Å³  3016.1(2) 1797.34(9) 1735.50(3) d_(calc), g/cm³2.494 2.359 2.252 R1(wR2)^(a) 0.0319 0.0253 0.0200 (0.0569) (0.0670)(0.0640) largest diff. 1.639 and −0.894 1.170 and −0.842 0.848 and−1.208 peak/hole (e Å⁻³) ^(a)[I > 2sigma(I)]

Example 2 Biological Properties

CO-releasing properties of cis-trans-[Re^(II)(CO)₂Br₂L₂]^(n) complexes.

The carbon monoxide releasing properties ofcis-trans-[Re^(II)(CO)₂Br₂L₂]^(n) complexes were evaluated by themyoglobin assay (FIG. 2A). This assay has been shown to be a reliablemethod for assessing the amount and kinetic of CO liberation fromCO-releasing molecules (Clark et al., Circ. Res. 2003, 93, e2-8,Motterlini et al., Circ. Res. 2002, 90, E17-24, Motterlini et al., Curr.Pharm. Des. 2003, 9, 2525-2539). In these experiments an aliquot of afreshly prepared concentrated solution of the [Re^(II)(CO)₂Br₂L₂]^(n)complex was added to a buffered solution of horse skeletal myoglobin(Mb) freshly reduced with excess sodium dithionite under N₂. With theexception of compound 2 all complexes tested are insoluble in water.However, stock solutions of 2 in water were found to generate aninactive form of the complex due to the accelerated CO release from thiscompound. Thus, solutions of 2 were prepared in methanol while stocksolutions of compounds 3-13 were prepared in DMSO. The final methanol orDMSO content of the buffered aqueous solution never exceeded 0.5%. Theconversion of Mb to carbon monoxide myoglobin (MbCO) was followed overtime by measuring the changes in the absorption spectra of the Q bandregion of this protein at pH 7.4, 6.3 and 5.8 after addition of theRe^(II) complex. The maximal absorption peak of Mb at 560 nm was rapidlyconverted over time to spectrum of MbCO, with two maximal absorptionpeaks at 540 and 578, respectively. A typical spectrum is shown in FIG.1.

Only compounds bearing monodentate ligands (i.e. compounds 2-7 in Scheme1, compounds 16-20 in Scheme 2 and B₁₂-CORM-2 and B₁₂-CORM-4. in Scheme3) elicited the spectral changes associated with CO release. The amountof MbCO formed over time after addition of the Re complex to the Mbsolution was calculated according to the known extinction coefficients.The MbCO concentration was directly related to the equivalents of COreleased from the compounds and these were plotted as a function oftime. FIGS. 2 to 7 show the graphic representation of results relatingto the pH-dependent rate of CO release of differentcis-trans-[Re^(II)(CO)₂Br₂L₂]^(n) complexes. The half-lives (t_(1/2)) ofCO release from the Re^(II) complexes at different pH's were estimatedfrom these graphs and are listed in table 3.

TABLE 3 Half-lifes (t½, min, 25° C.)a for the release of 1 equivalent ofCO by cis- [Re^(II)(CO)₂Br₂L₂]^(n) complexes at different pH's.^(b)Complex t_(1/2) at pH 5.8 t_(1/2) at pH 6.3 t_(1/2) at pH 7.4[Re(CO)₂Br₄]²⁻ (2) 1.0 2.5 5.7 [Re(CO)₂Br₂(Melm)₂] (3) 19.9 27.0 40.7[Re(CO)₂Br₂(Bzlm)₂] (4) 8.4 12.3 14.0 [Re(CO)₂Br₂(4-pic)₂] (5) 15.2 20.323.6 [Re(CO)₂Br₂(lm)₂] (6) 29.8 41.3 42.3 [Re(CO)₂Br₂(py)₂] (7) 9.7 10.217.2 [Et₄N][Re(CO)₂Br₂(4 Not yet Not yet ca. 20 picoline amine)₂] (15)measured measured ^(a)Half-lives (t_(1/2)) were estimated from thefitted curves shown in FIG. 5. ^(b)0.1M phosphate buffer. ^(c)n.d. = notdetermined; i.e. the complex does not release CO.

When the experiments were performed under conditions of a limitingamount of the metal complexes, taking into account the molar extinctioncoefficient of MbCO, it was found that approximately 1 mol of CO wasreleased per mole of the corresponding —[Re^(II)(CO)₂Br₂L₂]^(n) species.Thus, only one CO ligand is liberated from the metal complexes 2-7. TheCO detection via this assay proceeded as expected with three isosbesticpoints at 552, 567 and 587 nm clearly visible in the spectrum (see FIG.1). As described above, changes in the spectrum are indicative of theconversion of Mb to MbCO.

However, after full CO saturation of Mb the molar absorptivity in the Qband region of MbCO increased over time beyond the expected calculatedvalue. All three isosbestic points were lost. There was no furtherspectral change in terms of the position of the maximal absorption peaksat 540 and 578, but only an apparent increase in their molarabsorptivity. This was true for all [Re^(II)(CO)₂Br₂L₂]^(n) speciestested and for all pH's and for complex 15. The rate of CO loss fromcompounds 2-7 was found to be pH-dependent with half lives (t_(1/2))under physiological conditions (pH 7.4) varying from about 6 (for 2) to43 min (for 6, table 3). At lower pH values the time required to fullysaturate Mb with CO liberated from the metal complexes graduallydecreased. This was generally true for all compounds except for[Re(CO)₂Br₂(Im)₂] (6) and [Re(CO)₂Br₂(py)₂] (7), where only smalldifference were detected between pH 7.4 and 6.3 and between pH 6.3 and5.8 respectively (table 3).

Complex 2 was found to be the most rapid CO-releasing molecule (CORM) inall cases while the imidazole adducts were the slowest. The overallorder for the rate of CO release for the compounds tested is:2>4≈7>5>3≈6. At pH 7.4 and at 25° C. saturation of Mb with CO liberatedfrom compound 2 was reached within 30 min (t_(1/2)=5.7 min). This valueis comparable to that of the fac-[RuCl(glycinato)(CO)₃] complex (CORM-3)whose t_(1/2) at pH 7.4 and at 37° C. has been reported to be about 1min.

The above results prove that for the compounds of the invention, therate of CO release can be controlled by the appropriate choice ofligands. Thus, fine tuning of the coordination sphere allows for thedesign of inventive compounds with specific rates of CO loss.

Cytoprotective Effects of B₁₂-CORMs.

B₁₂-CORM-2 and 4 (Scheme 3) were tested for their cytotoxic and theircytoprotective effects using the neonatal rat cardiomyocyte (NRC)cell-based model of ischemia-reperfusion injury (I/R) as previouslydescribed (Zobi et al., Inorg. Chem. 2010, 49, 7313-7322). Themembrane-impermeable ReII-CORMs studied so far were non-toxic in themicromolar concentration range. To test for the possible uptake ofB₁₂—CO-RMs by cells incubation of NRCs with 30 μM of B₁₂-CORM-2 and 4was performed and cell culture medium samples were collected over 180min of incubation. Atomic absorption spectroscopy (AAS) measurementsshowed that the rhenium concentration in the medium supplemented withB₁₂-CORM-2 and B₁₂-CORM-4 did not change over time. This observationimplies that complexes (or the dissociated Re fragment) did not enterthe cells through the cell surface membrane during a 3 h incubationperiod (data not shown). The fraction of dead cells tended to decreasein the presence of CORMs, but, due to the high variability, thedifferences were not statistically significant (2.7±1.7% in control vs1.1±0.3 and 1.0±0.2 in the presence of B₁₂-CORM-2 and B₁₂-CORM-4respectively).

Exposure of the NRCs to the conditions mimicking ischemia-reperfusionresulted in a 5-fold increase in the number of dead cells (control inFIG. 8). Administration of 30 μM B₁₂-CORM-2 at the “onset ofreperfusion” nearly prevented cell mortality (cell death was reduce byca. 80% as compared to control) whereas 30 μM B₁₂-CORM-4 reduced celldeath by ca. 50%. Thus, B₁₂-CORM-2 proved to be more efficient inpreventing I/R-induced cell death than B₁₂-CORM-4. Therefore B₁₂-CORM-2was chosen for further investigation and compared to[Et₄N]₂[Re^(II)Br₄(CO)₂] (2) and the cis-trans-[Re^(II)(CO)₂Br₂(Im)₂]complex (6) that also exhibited substantial cytoprotective effects.(Zobi et al., Inorg. Chem. 2010, 49, 7313-7322.). All three compoundsrelease CO in aqueous solution, albeit at different rates, and with theexception of the lipophilic 6 are well soluble in water. Under normalphysiological conditions the compounds showed no cytotoxicity towardsNCRs up to a tested concentration of 120 μM.

Carbon monoxide is known to suppress respiration in different cell typesby inhibiting the mitochondrial electron transfer chain and byinterfering with oxygen binding. Therefore, the effect of the threeselected CO-RMs on oxygen consumption by NRCs was investigated. None ofthe tested compounds displayed any significant effect on oxygenconsumption by NRCs within the concentration range relevant forcytoprotection within 30 min of observation. The lack of effect neitherdepended on the rate of CO release nor on the compound solubility.Interestingly, addition of 2 and to a lesser extent of B₁₂-CORM-2resulted in deoxygenation of the cell-free medium within minutes afteradministration. This CORM-induced deoxygenation was dose-dependent. Thedeoxygenation efficiency followed the kinetics of CO release (and thusthe decay of the Re(CO)₂ fragment) and was maximal for thefast-releasing 2 compound (FIG. 9A).

Oxidative stress triggered by acute hyperoxygenation is a hallmark ofreperfusion injury. Based on the findings presented in FIG. 9A selectedCORMs may be viewed as oxygen scavengers which, when applied early onduring reperfusion, may reduce the cellular oxidative damage. To testthis hypothesis intracellular reduced glutathione (GSH) levels weredetermined in cells 10 min after the “onset of reperfusion” in thepresence or absence of 30 μM 2, 6 and B₁₂-CORM-2. As shown in FIG. 9B,presence of 2 complex at reperfusion resulted in an increase in theintracellular reduced glutathione (GSH) levels reflecting itsantioxidative action. (FIG. 9B). This acute antioxidative effect of 2most likely reflects the ability of the intermediates formed upon COrelease to react with oxygen forming ReO₄ ⁻.

Although the molecular mechanisms of the observed cytoprotective effectsof the above-mentioned CO-RMs remain unknown, their cytoprotectiveaction may, at least in part, be attributed to the extracellular releaseof CO and to the de-oxygenating effect described above. Finally, ourobservations suggest that the protective effect of Re^(II)-based CO-RMsis not related to a decrease in mitochondrial respiration and secondaryfree radical production.

1. A method for the prophylaxis and/or treatment of diseases and/ormedical condition involving hypoxic, anoxic and/or mammalian inflamedtissue, the method comprising the administration to a mammal in needthereof or the ex-vivo administration to a tissue before grafting into amammal in need thereof of a therapeutically effective amount of at leastone compound of Formula (I):

wherein Re is Re(I) or Re(II); n is selected from 2⁻, 1⁻, 0, 1⁺ and 2⁺;L₁, L₂, L₃ and L₄ are pharmaceutically acceptable monodentate ligandscomprising either at least one heteroatom or at least one carbon-carbondouble or triple bond for binding the rhenium metal of the complex; or atautomer, an isomeric form, a racemate, a single enantiomer, adiastereomer or mixtures thereof, pharmaceutically acceptable saltthereof, prodrug thereof or an effective amount of a pharmaceuticalcomposition thereof.
 2. The method according to claim 1, wherein thedisease and/or medical condition is selected from the group consistingof cardiovascular diseases, ischemia-reperfusion injury, inflammatorydiseases, traumatic injury, transplant rejection, platelet aggregationand/or monocyte activation; neuron degeneration of the nervous system,radiation damage, cancer, penile erectile dysfunction, adult respiratorydistress syndrome, and disorders of the circadian rhythm.
 3. The methodaccording to claim 1, wherein the cardiovascular disease is selectedfrom cardiac hypoxia, cardiac infarction, cardiac hypertrophy,arteriosclerosis and hypertension.
 4. The method according to claim 1,wherein the inflammatory disease is selected from asthma and angina. 5.The method according to claim 1, wherein the traumatic injury isselected from a brain, kidney or liver injury.
 6. The method accordingto claim 2, wherein the disease and/or medical condition isischemia-reperfusion injury.
 7. The method according to claim 1, whereinthe monodentate ligands comprise a heteroatom selected from N, O, S andP.
 8. The method according to claim 1, wherein the compound of Formula(I) wherein Re is Re (II).
 9. The method according to claim 1, whereinthe compound of Formula (I) wherein at least one of L₁, L₂, L₃ and L₄ isindependently selected from the group consisting of alkyl and cycloalkylcomprising at least one heteroatom, alkenyl, alkynyl, alkylidene, aryl,heteroaryl, arylalkyl, aryloxy, alkoxy, alkylthio, acyl, alkoxycarbonyl,acyloxy, acylamino, sulphonylamino, aminosulfonyl, alkylsulfonyl,carboxy, carboxamide, hydroxyl, oxo, halogen, trifluoromethyl, nitro,nitrile, isocyanide, alcohol, phosphine, phosphite, phosphonite,sulphide, sulfoxide and amino or guanidine, each amino or guanidineoptionally mono-, di- or tri-substituted by alkyl, acyl oralkoxycarbonyl, each member of the group optionally substituted by oneto four R″; wherein each R″ is independently selected from alkyl,alkenyl, alkynyl, alkylidene, cycloalkyl, aryl, arylalkyl, aryloxy,alkoxy, alkylthio, acyl, alkoxycarbonyl, acyloxy, acylamino,sulphonylamino, aminosulfonyl, alkylsulfonyl, carboxy, carboxamide,hydroxy, halogen, trifluoromethyl, nitro, nitrile and amino optionallymono-, di- or tri-substituted by alkyl, acyl or alkoxycarbonyl, whereinany members of the group and/or R″ are optionally halogenated wherepossible.
 10. The method according to claim 1, wherein the compound ofFormula (I) is of formula [Re^(II)Br₂(CO)₂(L)₂] wherein at least atleast one L is selected from the group consisting of halides, carbonmonoxide, N-methyl imidazole, benzimidazole, 4-methyl pyridine,imidazole, pyridine, C₁₋₆ alkyl cyanide, and alcohol, optionallysubstituted by one to four R″ where possible; wherein each R″ isindependently selected from the group consisting of alkyl, alkenyl,alkynyl, alkylidene, cycloalkyl, aryl, heteroaryl, arylalkyl, aryloxy,alkoxy, alkylthio, acyl, alkoxycarbonyl, acyloxy, acylamino,sulphonylamino, aminosulfonyl, alkylsulfonyl, carboxy, carboxamide,hydroxy, halogen, trifluoromethyl, nitro, nitrile and amino optionallymono-, di- or tri-substituted by C₁₋₆ alkyl, C₁₋₆ acyl or C₂₋₇alkoxycarbonyl; wherein any members of the group and/or of the R″ areoptionally halogenated where possible.
 11. The method according to claim1, wherein the compound of Formula (I) is such that at least one of L₁,L₂, L₃ and L₄ is Vitamin B12 optionally substituted optionallysubstituted by one to four R″ where possible; wherein each R″ isindependently selected from alkyl, alkenyl, alkynyl, alkylidene,cycloalkyl, aryl, heteroaryl, arylalkyl, aryloxy, alkoxy, alkylthio,acyl, alkoxycarbonyl, acyloxy, acylamino, sulphonylamino, aminosulfonyl,alkylsulfonyl, carboxy, carboxamide, hydroxy, halogen, trifluoromethyl,nitro, nitrile and amino optionally mono-, di- or tri-substituted byC₁₋₆ alkyl, C₁₋₆ acyl or C₂₋₇ alkoxycarbonyl; wherein any of the R″ areoptionally halogenated where possible.
 12. The method according to claim1, wherein the compound of Formula (I) is selected from the followinggroup: [Re(CO)₂(Br)₄]²⁻, [Re^(II)(CO)₂Br₂(N-methylimidazole)₂],[Re^(II)(CO)₂Br₂(benzimidazole)₂], [Re^(II)(CO)₂Br₂(4-methylpicoline)₂], [Re^(II)(CO)₂Br₂(Imidazole)₂], [Re^(II)(CO)₂Br₂(pyridine)₂]and [Et₄N][Re(CO)₂Br₂(4-picoline amine)₂].
 13. The method according toclaim 1, wherein the compound of Formula (I) is selected from thefollowing group: (Et₄N)₂[Re₂Br₆(CO)₄(pyrazine)],(Et₄N)₂[Re₂Br₆(CO)₄(pyrimidine)] and(Et₄N)₂[Re₂Br₆(CO)₄(5-Br-pyrimidine)].
 14. The method according to claim11, wherein the compound of Formula (I) is selected from the followinggroup:


15. The method according to claim 1, wherein the ligands L1-4 are in acis-trans-cis stereochemistry.
 16. The method according to claim 1,wherein the compound of Formula (I) or a tautomer, an isomeric form, aracemate, a single enantiomer, a diastereomer or mixtures thereof,pharmaceutically acceptable salt thereof, prodrug thereof or aneffective amount of a pharmaceutical composition thereof is administeredex-vivo in a vein graft before grafting.